CN104049082B - Human tissue kallikrein activity detection kit and application thereof - Google Patents
Human tissue kallikrein activity detection kit and application thereof Download PDFInfo
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- 230000000694 effects Effects 0.000 title claims abstract description 47
- 101000605522 Homo sapiens Kallikrein-1 Proteins 0.000 title claims abstract description 26
- 238000001514 detection method Methods 0.000 title claims abstract description 21
- 101710176219 Kallikrein-1 Proteins 0.000 claims abstract description 79
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 26
- 210000002700 urine Anatomy 0.000 claims abstract description 20
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 12
- YQDHCCVUYCIGSW-LBPRGKRZSA-N ethyl (2s)-2-benzamido-5-(diaminomethylideneamino)pentanoate Chemical compound NC(=N)NCCC[C@@H](C(=O)OCC)NC(=O)C1=CC=CC=C1 YQDHCCVUYCIGSW-LBPRGKRZSA-N 0.000 claims abstract 7
- 102000057032 Tissue Kallikreins Human genes 0.000 claims abstract 6
- 239000000243 solution Substances 0.000 claims description 51
- 238000002965 ELISA Methods 0.000 claims description 45
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 33
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 19
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 claims description 17
- 239000012530 fluid Substances 0.000 claims description 17
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 12
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 12
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 12
- 238000010790 dilution Methods 0.000 claims description 11
- 239000012895 dilution Substances 0.000 claims description 11
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 10
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 10
- 230000004927 fusion Effects 0.000 claims description 9
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 9
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 6
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 5
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- 239000008363 phosphate buffer Substances 0.000 claims description 5
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 5
- 239000001103 potassium chloride Substances 0.000 claims description 5
- 235000011164 potassium chloride Nutrition 0.000 claims description 5
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 5
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- 239000000126 substance Substances 0.000 abstract 1
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- 102100038297 Kallikrein-1 Human genes 0.000 description 74
- 241000283973 Oryctolagus cuniculus Species 0.000 description 16
- 208000006011 Stroke Diseases 0.000 description 10
- 206010008190 Cerebrovascular accident Diseases 0.000 description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 6
- 238000013016 damping Methods 0.000 description 6
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- 239000004793 Polystyrene Substances 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
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- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 230000001631 hypertensive effect Effects 0.000 description 1
- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 description 1
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- 230000002227 vasoactive effect Effects 0.000 description 1
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- 239000000052 vinegar Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
- G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
- G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
- G01N2333/96433—Serine endopeptidases (3.4.21)
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- Urology & Nephrology (AREA)
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- Biochemistry (AREA)
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses a human tissue kallikrein activity detection kit, which is characterized in that: the kit comprises: 1) coating a specific anti-human tissue kallikrein clone antibody enzyme label plate; 2) a human tissue kallikrein fusion protein TK-4T1 standard substance; 3) BAEE working solution. The kit adopts a method of combining immunity and chemistry, separates TK in a human urine or blood plasma sample from the sample by utilizing the characteristic that the TK can be combined with a specific antibody of the TK, and further catalyzes a substrate, namely benzoyl arginine ethyl ester hydrochloride (BAEE) to carry out chemical reaction.
Description
Technical field
The present invention relates to human tissue kallikrein detection field, refer to a kind of human tissue kallikrein activity detection kit and application thereof particularly, for detecting the activity of human tissue kallikrein.
Background technology
Human tissue kallikrein (TK) is a class serine protease, can promote that low molecule kininogen discharges vasoactive peptide thus plays a series of biological action, called after KLK1.In the model of the hypertension of humans and animals, angiocardiopathy and kidney trouble, all can see the reduction of TK content.We find that in genetically modified animal model TK has and reduce blood pressure, and improve insulin resistance, improve the effect of renal function.The level of TK may have important predictive value in the process of the angiocardiopathy of people and the generation development of kidney trouble.
We study discovery, and the generation of the content of TK and cerebral apoplexy (in ischemic cerebral apoplexy and hemorrhagic apoplexy) is the negative correlativing relation of dose response, and this phenomenon can exist independent of traditional risk factors of cardiovascular and cerebrovascular disease.Therefore, TK may be one of apoplexy patient independently protective factors.We followed up a case by regular visits to middle discovery to 5 years that all apoplexy patients carry out subsequently, and the patient that blood plasma TK content is higher has lower Stroke Recurrence rate and longer Event-free survival time.This illustrate TK not only with the generation of cerebral apoplexy about effect prevent Stroke Recurrence also may be had, and the predictor that the TK content of reduction also may recur as cerebral apoplexy.
Research finds that the gene pleiomorphism of TK promoter region and the reduction of TK activity and the centripetal reconstruction of artery are closely related.Therefore, the hereditary variation of TK gene may affect the expression of TK, activity and function and changes.In having bibliographical information to urinate the activity of TK and the hereditary variation of TK gene and hypertensive generation closely related.TK in urine or blood plasma is active there is no correlative study report with the relation of cerebral apoplexy and other cardiovascular and cerebrovascular diseases.And due to the complicated component in blood plasma, be unfavorable for the activity of direct-detection TK, so be necessary to set up a kind of kit of TK activity in blood plasma and preparation method thereof that can quantitatively detect, thus solve the complicated difficult problem being unfavorable for TK Activity determination of protein ingredient in blood plasma.
Summary of the invention
Technical matters to be solved by this invention is just to provide a kind of human tissue kallikrein activity detection kit and application thereof, this kit can catalytic substrate BAEE hydrochloride (BAEE) characteristic that chemical reaction occurs be combined by the characteristic that utilizes TK and can react with specific antibody and TK, thus solves protein ingredient complexity in blood plasma and be unfavorable for a difficult problem for TK Activity determination.
For solving the problems of the technologies described above, a kind of human tissue kallikrein activity detection kit provided by the invention, this kit comprises:
1) bag is by specific anti-human tissue kallikrein polyclonal antibody ELISA Plate;
2) human tissue kallikrein fusion TK-4T1 standard items;
3) BAEE working fluid,
Wherein, wrapping by the antibody of the anti-TK in the clonal antibody ELISA Plate of specific anti-human tissue kallikrein is the monoclonal antibody of anti-11P or the polyclonal antibody of anti-11P.Research shows: antigen has different epitopes, and different epitopes, in conjunction with different antibody, has different epitopes for human tissue kallikrein (TK).When we utilize the antibody of existing four kinds of anti-TK to carry out TK in examination criteria product active as capture antibody preparation feedback plate, only have good to line relation for anti-for the rabbit of the anti-11P typical curve drawn as the reaction plate prepared during capture antibody.This illustrate only have resist prepared reaction plate can be combined with TK with anti-11P rabbit while can also react with BAEE.
Further, described kit also comprises: the sodium hydroxide solution that PBST dilutes cleansing solution, watery hydrochloric acid, N are 3.5N, volumetric molar concentration to be the ferric chloride solution of 0.11M and volumetric molar concentration be in the hydroxylamine hydrochloride solution of 0.2M any one or a few.
Again further, the volumetric molar concentration of described BAEE working fluid is 0.05M.
Again further, it is 1% that described PBST dilutes Tween-20 massfraction in cleansing solution, the massfraction of described watery hydrochloric acid is 13.73%, and the N of sodium hydroxide solution is 3.5N, the volumetric molar concentration of ferric chloride solution be 0.11M and volumetric molar concentration is the hydroxylamine hydrochloride solution of 0.2M.
Present invention also offers a kind of bag by the preparation method of specific anti-human tissue kallikrein polyclonal antibody ELISA Plate, comprise the following steps:
1) weighing sodium carbonate and sodium bicarbonate, obtains pH and is 9.6 and volumetric molar concentration is the carbonate buffer solution of 0.05 in solution water; Namely bag is buffered liquid:
2) be buffered with above-mentioned bag the clonal antibody to 3200 times that liquid dilutes anti-TK, obtain antibody-solutions;
3) ratio adding 100 μ l in each hole draws antibody-solutions, and is added by antibody-solutions in the ELISA Plate in 96 holes, wraps and spent the night under 4 DEG C of conditions, is then that the PBS liquid 200 μ l of 1% is in 37 DEG C of closed 1h with BSA massfraction;
4) potassium dihydrogen phosphate, 12 water sodium hydrogen phosphates, sodium chloride is taken.Potassium chloride and Tween-20 prepare the phosphate buffer PBST that Tween-20 massfraction is 0.1%;
5) by step 4) the PBST washing step 3 that obtains) in the ELISA Plate closed, with PBST washing 2 ~ 3 times, each 3 minutes, pat dry and seal, namely obtaining bag by specific anti-human tissue kallikrein polyclonal antibody ELISA Plate.
Present invention also offers kit at the application process detecting TK activity in blood plasma and urine: comprise the following steps:
1) weighing sodium carbonate and sodium bicarbonate, obtains pH and is 9.6 and volumetric molar concentration is the carbonate buffer solution of 0.05 in solution water; Namely bag is buffered liquid:
2) be buffered with above-mentioned bag the clonal antibody to 3200 times that liquid dilutes anti-TK, obtain antibody-solutions;
3) ratio adding 100 μ l in each hole draws antibody-solutions, and is added by antibody-solutions in the ELISA Plate in 96 holes, wraps and spent the night under 4 DEG C of conditions, is then that the PBS liquid 200 μ l of 1% is in 37 DEG C of closed 1h with BSA massfraction;
4) potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride is taken.Potassium chloride and Tween-20 prepare the phosphate buffer PBST that Tween-20 massfraction is 0.1%;
5) by step 4) the PBST washing step 3 that obtains) in the ELISA Plate closed, with PBST washing 2 ~ 3 times, each 3 minutes, pat dry and seal, namely obtaining bag by specific anti-human tissue kallikrein polyclonal antibody ELISA Plate;
6) by step 5) ELISA Plate that obtains hatches 24h under being placed in 4 DEG C of conditions, then closes 1h with the BSA solution that massfraction is 1%;
7) utilize PBST to dilute cleansing solution dilution standard product and sample, standard items, sample and blank after dilution added step 6 respectively) in the ELISA Plate closed, and react 60min under ELISA Plate being placed in 37 DEG C of conditions;
8) to step 7) ELISA Plate in add BAEE working fluid, under 37 DEG C of conditions, react 60min;
9) to step 8) in ELISA Plate in add the mixed liquor of NaOH and oxammonium hydrochloride, make it fully to react with the BAEE do not consumed;
10) to step 9) ELISA Plate in add watery hydrochloric acid cessation reaction after add ferric chloride solution colour developing;
11) by microplate reader reading and drawing standard curve, calculate the activity of TK simultaneously.
Principle of the present invention:
The method that kit of the present invention adopts immunity to combine with chemistry, it separates and makes it be adsorbed in polystyrene board by the characteristic utilizing the TK in human urine or plasma sample can be combined with its specific antibody from sample, then BAEE effect after being situated between by the vinegar of TK, remaining BAEE and oxammonium hydrochloride and ferric trichloride effect generate coloring matter, calculate the activity of TK in sample finally by microplate reader reading.
Beneficial effect of the present invention is:
Kit of the present invention has highly sensitive, and its sensitivity is 0.040Eu ~ 1.27Eu, high specificity, reproducible feature.Human tissue kallikrein fusion TK-4T1 standard items are prokaryotic expression and the human tissue kallikrein of purifying (TK-4T1 fusion); Utilize this fusion as standard items, the related coefficient of its typical curve is up to 0.993.The TK utilizing this kit to have detected in urine specimen is active, and comparing difference average in the group between its multiple hole is 2.7%, and the average of its difference in the plate that the same day, duplicate detection TK activity obtained afterwards is 3.8%.Interval activity of TK in these samples of duplicate detection again after 6 months, between its plate, difference is 6.9%.Between the plate that we utilize this kit to compare when urine specimen is kept at 4 DEG C and-80 DEG C respectively, difference is 4.7%.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE figure of human tissue kallikrein fusion TK-4T1;
Fig. 2 is the SDS-PAGE figure of anti-TK-4T1 recombinant protein polyclonal antibody (rabbit of anti-11P resists);
Fig. 3 is the ELISA Plate of the different antibodies bag quilt amount of BAEE and OD when detecting
450canonical plotting between value;
Fig. 4 is ELISA Plate examination criteria product and the OD of different antibodies bag quilt
450canonical plotting between value;
Fig. 5 detects TK activity difference figure in urine and blood specimen for using TK activity detection kit.
Embodiment
In order to explain the present invention better, illustrate main contents of the present invention further below in conjunction with specific embodiment, but content of the present invention is not only confined to following examples.
The preparation of human tissue kallikrein activity detection kit
1, reagent
1) bag is buffered liquid (pH=9.6/0.05 carbonate buffer solution):
2) PBST dilutes cleansing solution (pH=7.4/0.15M)
3) BAEE working fluid
Utilize the Tris-HCl damping fluid of pH=8.0 by the BAEE Dilution for powder of 1.7191g to 100ml, be 0.05MBAEE.
4) other reagent
Watery hydrochloric acid is converted into according to volume ratio 1:2 with water by concentrated hydrochloric acid (concentration about 37%), and its massfraction is 13.73%,
The N of sodium hydroxide solution is 3.5N, the volumetric molar concentration of ferric chloride solution be 0.11M and volumetric molar concentration is the hydroxylamine hydrochloride solution of 0.2M.
Human tissue kallikrein fusion TK-4T1 standard items are prokaryotic expression and the human tissue kallikrein of purifying (TK-4T1 fusion) (reference paper " Plasmatissuekallikreinlevelisnegativelyassociatedwithinc identandrecurrentstroke:Amulticentercase-controlstudyinC hina " and patent " preparation of ELISA kit of human tissue kallikrein ").
2. key instrument
(1) microplate reader: ELX800 Μ niversalMicroplatereader
(2) ELISA Plate: CorningincorporatedcostarR96WellEIA/RIAplate
3. kit is detecting the application process of TK activity in blood plasma and urine: comprise the following steps:
1) weighing sodium carbonate and sodium bicarbonate, obtains pH and is 9.6 and volumetric molar concentration is the carbonate buffer solution of 0.05 in solution water; Namely bag is buffered liquid:
2) be buffered with above-mentioned bag the clonal antibody to 3200 times that liquid dilutes anti-TK, obtain antibody-solutions;
3) ratio adding 100 μ l in each hole draws antibody-solutions, and is added by antibody-solutions in the ELISA Plate in 96 holes, wraps and spent the night under 4 DEG C of conditions, is then that the PBS liquid 200 μ l of 1% is in 37 DEG C of closed 1h with BSA massfraction;
4) potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride is taken.Potassium chloride and Tween-20 prepare the phosphate buffer PBST that Tween-20 massfraction is 0.1%;
5) by step 4) the PBST washing step 3 that obtains) in the ELISA Plate closed, with PBST washing 2 ~ 3 times, each 3 minutes, pat dry and seal, namely obtaining bag by specific anti-human tissue kallikrein polyclonal antibody ELISA Plate;
6) by step 5) ELISA Plate that obtains hatches 24h under being placed in 4 DEG C of conditions, then closes 1h with the BSA solution that massfraction is 1%;
7) utilize PBST to dilute cleansing solution dilution standard product and sample, standard items, sample and blank after dilution added step 6 respectively) in the ELISA Plate closed, and react 60min under ELISA Plate being placed in 37 DEG C of conditions;
8) to step 7) ELISA Plate in add BAEE working fluid, under 37 DEG C of conditions, react 60min;
9) to step 8) in ELISA Plate in add the mixed liquor of NaOH and oxammonium hydrochloride, make it fully to react with the BAEE do not consumed;
10) to step 9) ELISA Plate in add watery hydrochloric acid cessation reaction after add ferric chloride solution colour developing;
11) by microplate reader reading and drawing standard curve, calculate the activity of TK simultaneously.
4. verification method
The anti-preparation of rabbit of 4.1 4 kinds of antibody
The rabbit of 4.11 anti-11P resists and the rabbit of anti-12P resists (reference paper " preparation of the bioinformatic analysis of human tissue kallikrein and the polyclonal antibody of different epitope thereof and purifying " and patent " a kind of monoclonal antibody of anti-human tissue kallikrein and preparation thereof ");
The rabbit of 4.12 anti-TK-4T1 resists and the monoclonal antibody of TK (reference paper " Plasmatissuekallikreinlevelisnegativelyassociatedwithinc identandrecurrentstroke:Amulticentercase-controlstudyinC hina " and patent " preparation of ELISA kit of human tissue kallikrein ").
In this enforcement, use rabbit to produce polyclonal antibody, in real process, other animals can also be selected to produce polyclonal antibody, as small white mouse etc.
4.2 detect the TK of human tissue kallikrein fusion TK-4T1 standard items active time the amount of BAEE and OD
450relation between value
(1) prepare by this laboratory for the various polyclonal antibody of the different epi-position of TK and monoclonal antibody (rabbit of anti-11P resists and the rabbit of anti-12P resists, and the rabbit of anti-TK-4T1 resists, the monoclonal antibody of TK);
(2) the various antibody of anti-TK are buffered liquid with bag respectively and are diluted to 3200 times;
(3) draw the various antibody 100 μ l of anti-TK respectively, joined in 96 hole ELISA Plate and spend the night in 4 DEG C;
(4) massfraction be 1% BSA close 1 hour, with PBST dilute cleansing solution wash 3 times;
(5) the BAEE working fluid of 0,10,20,40,60,80 μ l is drawn respectively in ELISA Plate;
(6) Tris-Hcl to the 100 μ l of 0.025MpH8.0 is added respectively;
(7) ELISA Plate is inserted in 37 DEG C of incubators and hatch 1 hour;
(8) ELISA Plate is placed on ice, adds 3.5N NaOH (50 μ l) and the mixed liquor 100 μ l of 0.2M oxammonium hydrochloride (50 μ l) in every hole respectively, mix rear room temperature and leave standstill 15 minutes;
(9) add watery hydrochloric acid 50 μ l, add 0.11M iron chloride 100 μ l after mixing, room temperature reaction 30 minutes;
(10) its 450nm wavelength place absorbance is surveyed by microplate reader, and drawing standard curve.
The selection of 4.3 best coated antibodies
(1) prepare by this laboratory for the various polyclonal antibody of the different epi-position of TK and monoclonal antibody (rabbit of anti-11P resists and the rabbit of anti-12P resists, and the rabbit of anti-TK-4T1 resists, the monoclonal antibody of TK).
(2) the various antibody of anti-TK are buffered liquid dilution with 0.05MpH9.6 carbonate bag respectively and cause 3200 times;
(3) draw the various antibody 100 μ l of anti-TK respectively, joined in 96 hole ELISA Plate and spend the night in 4 DEG C;
(4) massfraction be 1% BSA close 1 hour, with PBST dilute cleansing solution wash 3 times;
(5) standard items (TK-4T1) PBS is diluted to 1.00 μ g/ml, then doubling dilution to 0.031 μ g/ml is carried out to it;
(6) the standard items 100 μ l drawing each concentration respectively, in ELISA Plate, fully reacts 1 hour in 37 DEG C, washes plate 3 times;
(7) amount and the OD that draw reflection BAEE is selected
450tris-Hcl damping fluid and BAEE working fluid is added respectively: 100 μ l+0,90 μ l+10 μ l, 80 μ l+20 μ l, 60 μ l+40 μ l, 40 μ l+60 μ, 80 μ l+20 μ l in the hole of value typical curve.The Kong Ze of standard items is had to add Tris-Hcl damping fluid 40 μ l, BAEE working fluid 60 μ l; 37 DEG C are fully reacted 1 hour;
(8) add the mixed liquor 100 μ l of NaOH and oxammonium hydrochloride, make it fully to react 15 minutes with the BAEE do not consumed;
(9) FeCl is added after adding watery hydrochloric acid 50 μ l cessation reaction
3colour developing;
(10) utilize the antibody of existing four kinds of anti-TK to carry out the activity of TK in examination criteria product as coated antibody preparation feedback plate respectively: respectively with the value of standard items 1.00 μ g/ml, 0.500 μ g/ml, 0.250 μ g/ml, 0.125 μ g/ml, 0.063 μ g/ml, 0.031 μ g/ml for horizontal ordinate, the OD value of its correspondence is active for ordinate drawing standard curve calculates variable concentrations standard items TK simultaneously, Eu=(T-M)/t.(Eu: be TK active unit, namely in the Tris-Hcl damping fluid of 0.025MpH8.0, is an enzyme unit when the amount of the BAEE of 37 DEG C of hydrolysis per minute l μm of ol; T: the total amount (μm ol) adding BAEE; M: the amount namely directly recorded by the amount (μm ol) of remaining BAEE in reactant liquor after TK effect; T: incubation time (dividing).
4.4 sample determinations (method is in table 3)
(1) get the sample to be tested of 10 μ l, be diluted to 100 μ l with PBST;
(2) 12 holes of reserved typical curve add (comprising multiple hole) PBST of 100 μ l, remaining hole adds sample (20 routine plasma samples and 20 routine urine samples) dilution 100 μ l respectively, fully react 1 hour in 37 DEG C, wash plate 3 times;
(3) 12 holes of doing typical curve are selected to add Tris-Hcl damping fluid 100 μ l, 90 μ l, 80 μ l, 60 μ l, 40 μ l and 20 μ l respectively; Remaining sample well all adds 40 μ l;
(4) sample well adds the BAEE of 60 μ l respectively, and 37 DEG C are fully reacted 1 hour;
(5) add the mixed liquor 100 μ l of NaOH and oxammonium hydrochloride, make it fully to react 15 minutes with the BAEE do not consumed;
(6) FeCl is added after adding the watery hydrochloric acid cessation reaction of 50 μ l
3colour developing;
(7) by microplate reader reading and drawing standard curve, calculate the activity of TK, Eu=(T-M) * 1000/tv simultaneously.(Eu: be TK active unit, namely in the Tris-Hcl damping fluid of 0.025MpH8.0, is an enzyme unit when the amount of 37 DEG C of hydrolysis per minute l μm of olBAEE; T: the total amount (μm ol) adding BAEE; M: the amount namely directly recorded by the amount (μm ol) of remaining BAEE in reactant liquor after TK effect; T: incubation time (dividing); V: the urine volume (μ l) that timing is used).
The Quality Identification of 4.5 kits
(1) sensitivity of kit: the TK activity of drawing after carrying out doubling dilution according to standard items and OD
450the sensing range of this kit that the typical curve of relation calculates between value.
(2) comparison of group difference and the interior difference of group: the TK detected respectively in 20 routine blood plasma and urine sample liquid is active, wherein often group does three multiple holes, and in duplicate detection on the same day.Then difference and group difference in its group is compared.
(3) Detection of Stability of kit: by finished product kit in 4 DEG C of preservations, and after 6th month, its quality is detected.
(4) detection of sample storage condition: newly collect 15 routine urine specimens are stored in 4 DEG C and-80 DEG C respectively, detects the activity of TK in urine next day.
4.6 result
4.6.1 the amount of BAEE and OD when the TK of table 1 examination criteria product is active
450relation between value
The antibody of existing four kinds of anti-TK is utilized to carry out the activity of TK in examination criteria product as capture antibody preparation feedback plate, with 0, value corresponding to 0.005M, 0.01M, 0.02M, 0.03M, 0.04MBAEE be for horizontal ordinate, the OD value of its correspondence is ordinate drawing standard curve, and these four typical curves are all to line good (table 1 Fig. 3).
4.6.2 the active OD of the TK of table 2 standard items
450relation between value
When with the concentration of standard items TK-4T1 for horizontal ordinate, when the OD value of its correspondence is ordinate drawing standard curve, only has the typical curve anti-for the rabbit of anti-11P (anti-11P) drawn as the reaction plate prepared during capture antibody to line relation good (r=0.990, table 2 Fig. 4).This illustrate only have could be combined with TK with the reaction plate prepared by anti-11P rabbit anti-(anti-11P) while can also react with BAEE.
4.6.3 table 3TK Activity determination
When table 3 points out end user tissue kallikrein activity detection kit monitor sample TK active, the Adding Way of the gauge orifice of production standard curve and each reagent of sample aperture of detection sample.
4.6.4 blood plasma and urine in TK Activity determination
The TK that have detected respectively in 20 routine plasma samples and urine is active, found that the activity of TK in urine is 1.11 ± 0.53Eu/ml; In blood plasma, TK activity is 2.28 ± 0.61Eu/ml.TK in blood plasma is active in urine TK activity (P<0.001; Fig. 5).
4.6.5 the Quality Identification of kit
The sensitivity of kit of the present invention is 1.271Eu ~ 0.0401Eu.The TK utilizing this kit to have detected in 20 routine urine specimens is active, and comparing difference average in the group between its multiple hole is 2.7%, and in the plate that the same day, duplicate detection TK activity obtained afterwards, the average of difference is 3.8%.Interval activity of TK in these samples of duplicate detection again after 6 months, between its plate, difference is 6.9%.Between the plate that we utilize this kit to compare when 15 routine urine specimens are kept at 4 DEG C and-80 DEG C respectively, difference is 4.7%.
Other unspecified part is prior art.Although above-described embodiment is to invention has been detailed description; but it is only the present invention's part embodiment; instead of whole embodiment, people can also obtain other embodiments according to the present embodiment under without creative prerequisite, and these embodiments all belong to scope.
Claims (4)
1. a human tissue kallikrein activity detection kit, is characterized in that: this kit comprises:
1) bag is by specific anti-human tissue kallikrein clonal antibody ELISA Plate;
2) human tissue kallikrein fusion TK-4T1 standard items;
3) BAEE working fluid;
Described kit also comprises: PBST to dilute in cleansing solution, watery hydrochloric acid, sodium hydroxide solution, ferric chloride solution and hydroxylamine hydrochloride solution any one or a few;
Wherein, wrapping by the clonal antibody of the anti-TK in specific anti-human tissue kallikrein clonal antibody ELISA Plate is the monoclonal antibody of anti-11P or the polyclonal antibody at least containing anti-11P, and the volumetric molar concentration of described BAEE working fluid is 0.05M.
2. human tissue kallikrein activity detection kit according to claim 1, it is characterized in that: it is 1% that described PBST dilutes Tween-20 massfraction in cleansing solution, the massfraction of described watery hydrochloric acid is 13.73%, and the N of sodium hydroxide solution is 3.5N, the volumetric molar concentration of ferric chloride solution be 0.11M and volumetric molar concentration is the hydroxylamine hydrochloride solution of 0.2M.
3. bag is by a preparation method for specific anti-human tissue kallikrein polyclonal antibody ELISA Plate, it is characterized in that: comprise the following steps:
1) weighing sodium carbonate and sodium bicarbonate, obtains pH and is 9.6 and volumetric molar concentration is the carbonate buffer solution of 0.05 in solution water; Namely bag is buffered liquid:
2) be buffered with above-mentioned bag the clonal antibody to 3200 times that liquid dilutes anti-TK, obtain antibody-solutions;
3) ratio adding 100 μ l in each hole draws antibody-solutions, and is added by antibody-solutions in the ELISA Plate in 96 holes, wraps and spent the night under 4 DEG C of conditions, is then that the PBS liquid 200 μ l of 1% is in 37 DEG C of closed 1h with BSA massfraction;
4) take potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride and Tween-20 and prepare the phosphate buffer PBST that Tween-20 massfraction is 0.1%;
5) by step 4) the PBST washing step 3 that obtains) in the ELISA Plate closed, with PBST washing 2 ~ 3 times, each 3 minutes, pat dry and seal, namely obtaining bag by specific anti-human tissue kallikrein polyclonal antibody ELISA Plate.
4. the kit described in claim 1 ~ 2 any one is detecting an application for TK activity in blood plasma and urine, comprises the following steps:
1) weighing sodium carbonate and sodium bicarbonate, obtains pH and is 9.6 and volumetric molar concentration is the carbonate buffer solution of 0.05 in solution water; Namely bag is buffered liquid:
2) be buffered with above-mentioned bag the clonal antibody to 3200 times that liquid dilutes anti-TK, obtain antibody-solutions;
3) ratio adding 100 μ l in each hole draws antibody-solutions, and is added by antibody-solutions in the ELISA Plate in 96 holes, wraps and spent the night under 4 DEG C of conditions, is then that the PBS liquid 200 μ l of 1% is in 37 DEG C of closed 1h with BSA massfraction;
4) take potassium dihydrogen phosphate, 12 water sodium hydrogen phosphates, sodium chloride, potassium chloride and Tween-20 and prepare the phosphate buffer PBST that Tween-20 massfraction is 0.1%;
5) by step 4) the PBST washing step 3 that obtains) in the ELISA Plate closed, with PBST washing 2 ~ 3 times, each 3 minutes, pat dry and seal, namely obtaining bag by specific anti-human tissue kallikrein polyclonal antibody ELISA Plate;
6) by step 5) ELISA Plate that obtains hatches 24h under being placed in 4 DEG C of conditions, then closes 1h with the BSA solution that massfraction is 1%;
7) utilize PBST to dilute cleansing solution dilution standard product and sample, standard items, sample and blank after dilution added step 6 respectively) in the ELISA Plate closed, and react 60min under ELISA Plate being placed in 37 DEG C of conditions;
8) to step 7) ELISA Plate in add BAEE working fluid, under 37 DEG C of conditions, react 60min;
9) to step 8) in ELISA Plate in add the mixed liquor of NaOH and oxammonium hydrochloride, make it fully to react with the BAEE do not consumed;
10) to step 9) ELISA Plate in add watery hydrochloric acid cessation reaction after add ferric chloride solution colour developing;
11) by microplate reader reading and drawing standard curve, calculate the activity of TK simultaneously.
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| CN105985436B (en) * | 2015-01-30 | 2019-04-12 | 江苏众红生物工程创药研究院有限公司 | Anti-human kininogenase antibody and its application |
| CN105987998B (en) * | 2015-01-30 | 2017-12-29 | 江苏众红生物工程创药研究院有限公司 | Human tissue kallikrein 1ELISA immue quantitative detection reagent boxes |
| US20200174021A1 (en) * | 2017-08-08 | 2020-06-04 | Queensland University Of Technology | Methods for diagnosis of early stage heart failure |
| CN115478063A (en) * | 2022-09-20 | 2022-12-16 | 华中科技大学同济医学院附属同济医院 | Extraction and purification method of human tissue kallikrein |
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| CN101210926A (en) * | 2006-12-27 | 2008-07-02 | 中国科学院大连化学物理研究所 | Reagent kit for detecting human serum tissue kallikrein and its preparation method |
| CN101672851A (en) * | 2009-07-31 | 2010-03-17 | 华中科技大学同济医学院附属同济医院 | Preparation of Human Tissue Kallikrein ELISA Kit |
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| CN101210926A (en) * | 2006-12-27 | 2008-07-02 | 中国科学院大连化学物理研究所 | Reagent kit for detecting human serum tissue kallikrein and its preparation method |
| CN101672851A (en) * | 2009-07-31 | 2010-03-17 | 华中科技大学同济医学院附属同济医院 | Preparation of Human Tissue Kallikrein ELISA Kit |
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